Method for increasing agglutination of groups of cells to produce improved cell layer interface in centrifuged blood sample

ABSTRACT

In order to produce a more well defined interface between adjacent cell layers in a centrifuged sample of anticoagulated whole blood, a material which will bond one group of cells together is added to the blood sample prior to centrifugation. The bonding material must produce a high strength bond between one group of cells, but not effect the other cell types. The material is added prior to centrifugation of the blood sample.

This application is a continuation of application Ser. No. 794,127,filed Nov. 1, 1985, now U.S. Pat. No. 4,695,553.

This invention relates to a method for providing an improved betterclarified interface between the erythrocyte layer and the leukocytelayer in a centrifuged sample of anticoagulated whole blood.

U.S. Pat. No. 4,027,660 issued Apr. 2, 1976; and U.S. Pat. No. 4,082,085issued Apr. 4, 1978 relate to a technique for performing differentialleukocyte, counts in a sample of anticoagulated whole blood which hasbeen drawn into a capillary tube and centrifuged. A generallycylindrical float is disposed in the capillary tube in the blood sampleduring centrifugation. The float settles into the erythrocyte layer andextends through the buffy coat so as to physically elongate theleukocyte and platelet layers. A stain such as acridine orange is usedto differentially color the different constituents which make up thebuffy coat so that the buffy coat appears as a plurality of differentlycolored bands in the capillary tube. The layering constituents accordingto density during centrifugation allows cell counts to be made bymeasuring the distance between the boundaries of each cell band.

U.S. Pat. No. 4,159,896 issued July 3, 1979; and U.S. Pat. No. 4,181,609issued Jan. 1, 1980 describe a problem, and a solution therefor, whichis encountered in some blood samples tested in accordance with theabove-described technique. The problem is a poorly defined demarcationbetween the top of the erythrocyte layer and the bottom of the leukocytelayer. The poor definition occurs because abnormally low densityerythrocytes which are found in the blood sample tend to settle in thelower-most region of the leukocyte layer. The solution taught by thenoted prior art is to add potassium oxalate to the blood sample toincrease the density of the erythrocytes so that the less denseerythrocytes will sediment with the majority of cells in the erythrocytelayer during centrifugation. In order to accentuate the increaseddensity, the blood sample may be warmed while in the capillary tubeprior to centrifugation. Following the prior art procedure, satisfactorydelineation of the erythrocyte-leukocyte boundary is achieved inapproximately 98% of normal out-patient cases and in approximately 70%of the hospitalized patients. In view of the ineffectiveness of theprior art procedures in a significant number of cases, particularly inthe hospital environment, it is apparent that a more efficaciousprocedure is desirable so that good erythrocyte-leukocyte separationwould be achieved in all cases.

This invention relates to a method of achieving a clear demarcationbetween the erythrocytes and leukocytes in a centrifuged sample ofanticoagulated whole blood in virtually every case.

The improved erythrocyte-leukocyte interface demarcation is accomplishedby adding a material to the blood sample which will bond theerythrocytes together prior to centrifugation of the sample. Thematerial which is added to the blood sample must produce a high strengthbond between the erythrocytes, regardless of type, while not affectingthe cells in the buffy coat. By thus agglomerating the erythrocytesprior to centrifugation, the erythrocytes will sediment according totheir mean density when the sample is centrifuged. The rate ofsedimentation of agglomerated cells will also be faster. Thus, the lessdense microcytes and reticulocytes will not layer on top of the heaviererythrocytes, as they normally do and, therefore, will not blur theerythrocyte-leukocyte interface. The materials which are added to theblood sample are materials such as: antibodies for H red cell antigensand/or mixed A, B, H antigens; lectins (vegetable carbohydrates withantibody-like properties) such as that from Eulex Europus; andantibodies directed against universal erythrocyte antigens, particularlymonoclonal antibodies. The preferred material is any humanerythrocyte-specific monoclonal antibody which can agglutinate thetarget cells. Mixed lectins are also effective. Both of these materialsmay be combined to provide an effective additive.

The problem solved by the above-noted procedure is caused by intrusionof abnormally less dense erythrocytes into normal granulocytes. Theseabnormally less dense erythrocytes may be small fragments caused by poorcell formation or caused by mechanical or other damage, or may beswollen erythrocytes resulting from metabolic damage. They may also beimmature erythrocytes which are known to have a lower buoyant densitythan mature erythrocytes. These cells are a relatively small part of thenormal density population, but they may be of sufficient number to mixwith and disrupt the granulocyte layer. The procedure of this inventionresults in an irrevocable merging of these abnormally less denseerythrocyte materials with the normal erythrocyte population. Thematerial which is added can be characterized as a sort of "glue"specific only to erythrocytes and which converts all of the erythrocytesinto small aggregates of cells of average density. Since that averagedensity is greater than the density of the granulocytes, completeseparation between the erythrocytes and the granulocytes will occur. Theaggregates must not be too large to also trap leukocytes in theclusters.

When lectins are used as the agglutinating material, the followingapplies. Lectins such as Eulex Europus, Triticum Vulgaris, and PhaseolusVulgaris may be used in this procedure and are available in powder formfrom Sigma Chemical Company of St. Louis, Mo. It is well known that whenutilizing biologic material such as lectins, it is often impossible tospecify an effective solution concentration of the biologic material,since the active constituents of the material are not known, or, are atleast poorly defined. Thus, it is necessary to determine an effectiveconcentration of the respective lectins by proceeding with sequentialtitrations.

A known weight or volume of the lectin material (in powder form) isdissolved in a known volume of an appropriate medium, such as saline.The initial concentration is only important as a known starting pointfrom which an effective concentration is to be derived: Sequentialtitrations are then made from serial dilutions of the starting fluid.Half dilutions are recommended. This procedure is continued so as tocreate a range of concentrations from the original solution to the mostdilute operative solution. During the progression, several drops of eachdilution are mixed with an equal amount of the blood. The resultingmixtures are then examined by visual inspection for agglutination of theerythrocytes. When a minimum effective concentration or the mixture (atitre) is found, that concentration is enhanced a factor of about fourto ten so as to ensure effective agglutination of the erythrocytes andeconomize reagents. By using an excess in this manner, possibledegradation of the solution over time is negated.

As noted above, monoclonal antibodies which are specific to antigensfound on all human erythrocytes, but not on leukocytes, are a preferredagglutinating material for use in the procedure of this invention. Someantigens are found on virtually all human erythrocytes. Examples of sucha universal erythrocyte antigen are the H antigen and Glycophorin. Thepreparation and selection of anti-sera and monoclonal antibodies tothese types of antigen are well known High concentrations of monoclonalantibodies can be produced as ascites fluid using standard immunologicaltechniques. The antibody-containing ascites fluid is harvested from themouse, and it is diluted on a one to one thousand ratio with a bufferingagent. This produces the antibody-containing reagent which is used tocause the desired agglutination of the erythrocytes in the procedure ofthis invention.

Generally, the procedure of this invention is as follows. Theerythrocyte agglutinating material is dried on the inside of a capillarytube along with stains or dyes as necessary to visually differentiatethe cell layers. A sample of blood is into the coated capillary tube andthe float is positioned in the capillary tube. The bottom of the tube iscapped and the sample is centrifuged. A clear, evenerythrocyte-leukocyte interface is achieved with no erythrocyte materialrising into the leukocyte layers. The buffy coat constituents bandnormally so as to allow accurate measurement of band heights and therebycreate accurate counts.

It will be readily appreciated that the procedure of this invention willeliminate the prior art problem of poor separation between erythrocytesand leukocytes which occurs in a significant number of tests. Theadditives do not affect the leukocytes and produce an evenerythrocyte-leukocyte interface for accurate measurements. Theerythrocytes are not prevented from buoying the float in the normalmanner.

This invention can also be used to selectively agglutinated cellsub-populations to form well defined layers of such sub-populations in acentrifuged blood sample. For example, the reticulocyte sub-populationof the erythrocytes can be banded separately from the remainingerythrocytes by adding a monoclonal antibody which is specific totransferrin receptors which are found on the surface of thereticulocytes. By this method, a more accurate measurement of thereticulocyte count can be achieved. This is a useful tool in thedifferential diagnosis of anemia, and is very time-consuming to performby traditional methods.

The invention can likewise be used to agglutinate the eosinophilsub-population of leukocytes into a distinct band in the centrifugedblood sample by adding to the blood sample an antibody which is specificto the eosinophils. At present, the eosinophils are too dispersedthroughout the granulocyte layer to be readily measured with thecentrifugation procedure.

A further improvement includes the addition of a fluorescent material tothe antibody used for agglutinating the cell types. For example, if theanti-transferrin receptor antibody were joined to fluorescein, thereticulocytes would be both separated and differentiated from the massof the other erythrocytes.

Since many changes and variations of the disclosed embodiments of theinvention may be made without departing from the inventive concept, itis not intended to limit the invention otherwise than as required by theappended claims.

What is claimed is:
 1. A method of removing unwanted blood cell typesfrom an assay area in a sample, said method comprising the steps of:agglutinating said unwanted blood cell types into clumps by adding aneffective amount of an antibody to the sample which selectively causesincreased agglutination of the unwanted blood cell types; and displacingsaid clumps out of said assay area of the sample.